This application is focused on the enhancement of immune responses to HIV envelope proteins, and seeks to develop a new method to facilitate targeted expression of native immunogens in vivo, with the aim of advancing HIV vaccine design. The experiments proposed will examine whether candidate DNA accines can be targeted more efficiently to APCs, with special emphasis on blood-derived DCs, by conjugating them to an adenovirus protein (penton base) that binds to specific cellular integrins and participates in endosome lysis, or by linking them to Type 1 HIV (HIV-1) Tat-derived peptide, which undergoes efficient cellular import. In Aim 1, the applicants will test whether adenovirus penton base protein (ADPB), which contains RGD and LDV motifs found in Ad7 and Ad11, can be used to facilitate DNA delivery to cultured blood DCs. In Aim 2, modifications will be introduced into ADPB to selectively alter its ability to interact with cellular integrins. Known integrin-binding motifs will be substituted into ADPB, including the HIV-1 Tat RGD motif, and a high-affinity RGD motif selected by phage display. The ability of these modified ADPBs to target cultured DCs will be examined. In Aim 3, the investigators will examine whether a bifunctional peptide, containing the basic domain of HIV-1 Tat plus a poly-lysine tract, can be used to deliver exogenous DNA molecules to DCs. In vivo studies will be conducted in Aims 4 and 5. In Aim 4, experiments will be conducted to determine whether native ADPBs (Aim 1), mutant ADPBs (Aim 2) or Tat-derived peptides (Aim 3), when conjugated to a reporter plasmid (E. coli beta-galactosidase (b-Gal), or green fluorescent protein (GFP)), can produce more efficient expression of a reporter gene and enhanced targeting to DCs in mice. These reporter genes show improved expression after intradermal injection or gene gun delivery. The optimal delivery agents then will be conjugated to HIV-1 DNA vaccine encoding gp120. Finally, the optimal ADPB derivative (from Aims 1-4) will be conjugated to a candidate HIV-1 DNA vaccine, and its ability to engender anti-HIV-1 immune responses in vivo (mice) will be compared to that of the same DNA construct, when delivered by conventional means.